Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

Why do tubulin gene families lack diversity in flagellate/ciliate protists?

Summary Considerable amino acid sequence diversity is found among tubulin isotypes encoded by tubulin gene families in animal, higher plant, and fungal systems. In contrast, relatively little diversity is found among the isotypes produced by the gene families in a number of flagellate or ciliate protists. It is possible that proper assembly of the axoneme requires a homogeneous pool of tubulin subunits and that the axoneme thus provides a stringent selection against amino acid replacement substitutions among tubulin genes in these systems.     

Subcellular Localization of Functionally Differentiated Microtubules in Squid Neurons: Regional Distribution of Microtubule-Associated Proteins and β-Tubulin Isotypes

The subcellular localization of microtubule proteins in the neurons of squid (Doryteuthis bleekeri) was immunologically studied using monoclonal antibodies against the microtubule proteins. We found that (1) the squid neurons contained three kinds of high-molecular-weight microtubule-associated proteins [MAP A of approximately 300 kilodaltons (kD), MAP B of 260 kD, and axolinin of 260 kD] and two kinds of beta-tubulin isotypes (beta 1 and beta 2); (2) the cell body of the squid giant neuron contained MAP A, MAP B, and the two beta-tubulin isotypes (beta 1 and beta 2); (3) axolinin and the beta 1 isotype were present exclusively in the peripheral axoplasm of the giant axon; and (4) a small amount of axolinin, MAP A, and the beta 1 isotype was found in the insoluble aspect of the central axoplasm, whereas the soluble aspect of the central axoplasm contained an abundant amount of MAP A along with the modified form of the beta 1 isotype. The regional difference of the distribution of the microtubule protein components may explain the differences in stability among axonal microtubules. Microtubules in the soluble aspect of the central axoplasm are sensitive to any treatment with colchicine, cold temperature, and high ionic strength but those both in the insoluble aspect of the central axoplasm and in the peripheral axoplasm are highly insensitive to the treatment.     

Regulation of β-tubulin function and expression in Drosophila spermatogenesis

In this study we examined two aspects of β-tubulin function in Drosophila spermatogenesis: 1) β-tubulin structural requirements for assembly of different categories of microtubules and 2) regulatory requirements for production of the correct tubulin protein level. In normal Drosophila spermatogenesis, the testis-specific β2-tubulin isoform supports multiple microtubule functions. Our previous work showed that another Drosophila isoform, β3, cannot support spermatogenesis, whereas a carboxyl-truncated form of β2, β2ΔC, can at least to some extent provide all of β2′s normal functions, save one: β2ΔC cannot support organization of axonemal microtubules into the supramolecular architecture of the axoneme. Here, to test whether β2 carboxyl sequences can rescue the functional failure of the β3 isoform in spermatogenesis, we constructed a gene encoding a chimeric protein, β3β2C, in which β3 sequences in the carboxyl region are replaced with those of β2. Unlike either β3 or β2ΔC, β3β2C can provide partial function for both assembly of axonemal microtubules and their organization into the supramolecular architecture of the axoneme. In particular, the β2 carboxyl sequences mediate morphogenesis of the axoneme doublet tubule complex, including accessory microtubule assembly and attachment of spokes and linkers. However, our data also reveal aspects of β2-specific function that require structural features other than the primary sequence of the isotype-defining variable regions, the C terminus and the internal variable region. Tests of fecundity in males that co-express Δ2 and the chimeric Δ3Δ2C protein showed that in Drosophila there are differential requirements for sperm motility in the male and in the female reproductive tract. Since some aspects of microtubule function in spermatogenesis are sensitive to the tubulin pool size, we examined the mechanisms for control of tubulin protein levels in the male germ cells. We found that both Δ2-tubulin mRNA accumulation and protein synthesis are dependent on gene dose, and that the level of expression is regulated by 3′ noncoding sequences in the Δ2 gene. Our data show that the regulatory mechanisms that control tubulin pool levels in the Drosophila male germ line differ from those observed in cultured animal somatic cells. Finally, expression of transgenic constructs is consistent with early cessation of × chromosome expression in Drosophila spermatogenesis. © 1995 Wiley-Liss, Inc.     

Tubulin Carboxypeptidase/Microtubules Association can be Detected in the Distal Region of Neural Processes

The association of tubulin carboxypeptidase with microtubules has been demonstrated in crude brain extracts and in living non-nervous cells. Here, we studied this phenomenon in cultured brain cells. To determine the association of the enzyme with neural microtubules we isolated the cytoskeletons (detergent-extraction under microtubule-stabilizing conditions) and measured the content of Tyr, Glu, and 2 tubulin as a function of the in vitro incubation time of the cytoskeletons. The carboxypeptidase was found associated with microtubules in 2 days-cultured cells but not in 7 days-cultured cells. Quantitative analysis of digitized images after immunofluorescent staining revealed that detyrosination during the incubation of the cytoskeletons occurred preferentially in the distal regions of the neural processes. Prolonged taxol-treatment of the cells promoted higher detyrosination but Tyr tubulin was not depleted suggesting the existence of a subset of microtubules that has not associated carboxypeptidase and therefore cannot be detyrosinated even after prolonged taxol-treatment. This hypothesis was supported, although not conclusively, by additional experiments.     

Human P-glycoprotein is active when the two halves are clamped together in the closed conformation

The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the two homologous halves of P-gp is composed of a transmembrane domain (TMD) with six TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures show drug pumps in the open and closed conformations, where the drug-binding pocket and NBDs are open or closed at the cytoplasmic side, respectively. Although it has been postulated that drug substrates enter the drug-binding pocket in the open conformation, it is unknown if they can enter in the closed conformation. To determine this, we introduced cysteines into regions of TM3 (residues 175-178) and TM9 (residues 820-822) that extend into the cytoplasm and are 4 Å and 20 Å apart in the closed and open conformations, respectively. The 12 double cysteine mutants were then cross-linked with a short cross-linker, M1M (4 Å) at 0 °C to reduce thermal motion in the protein. Only mutant L175C/N820C was cross-linked. Cross-linking was not increased in the presence of ATP or drug substrates. Cross-linking increased its basal ATPase activity about 3-fold. Activity could be increased further by drug substrates such as verapamil and rhodamine B. These results suggest that P-gp in the membrane is in the closed conformation that has a high affinity for drug substrates.     

Increased levels of tyrosinated alpha-, beta(III)-, and beta(IV)-tubulin isotypes in paclitaxel-resistant MCF-7 breast cancer cells

Paclitaxel (PTX), the diterpene alkaloid, is a potent anti-cancer drug and is routinely used for the treatment of breast and ovarian cancers. The cellular targets of PTX are microtubules, which are composed of alpha- and beta-tubulin. Development of PTX resistance in patients has been a major problem associated with cancer chemotherapy. In an effort to get insight into this phenomenon of drug resistance, a PTX-resistant cell line from MCF-7 breast cancer cells has been generated. Western analysis of the cell extracts revealed that the resistant cells contain 2-fold higher amount of tyrosinated alpha-tubulin than those of the wild-type MCF-7 cells. Similar analyses of beta-tubulin with the isotype-specific monoclonal antibodies demonstrated that the PTX-resistant cells contain 2.5-fold higher amounts of beta(III) and 1.5-fold higher amount of beta(IV)-tubulin, while no difference was observed in the level of beta(I) isotype. These results demonstrate for the first time that PTX resistance is associated with an increase in the level of tyrosinated alpha-tubulin.     

Tyrosine phosphorylation of plant tubulin

Phosphorylation of αβ-tubulins dimers by protein tyrosine kinases plays an important role in the regulation of cellular growth and differentiation in animal cells. In plants, however, the role of tubulin tyrosine phosphorylation is unknown and data on this tubulin modification are limited. In this study, we used an immunochemical approach to demonstrate that tubulin isolated by both immunoprecipitation and DEAE-chromatography is phosphorylated on tyrosine residues in cultured cells of Nicotiana tabacum. This opens up the possibility that tyrosine phosphorylation of tubulin could be involved in modulating the properties of plant microtubules.     

Cloning of rat olfactory bulb tubulin tyrosine ligase cDNA: a dominant negative mutant and an antisense cDNA increase the proliferation rate of cells in culture

In this paper we describe the cloning of rat olfactory bulb tubulin tyrosine ligase (TTL) cDNA, and investigate the physiological role of TTL in cultured CHO-K1 cells. Comparison of the deduced amino acid sequence of rat TTL cDNA with those of bovine and pig showed approximately 90% of identity. Transient transfection of CHO-K1 cells with a dominant negative mutant of TTL that contains the binding site to the substrate (tubulin) but not the catalytic domain, significantly decreased the endogenous TTL activity as determined in vitro. Similar results were obtained using a construction encoding for the antisense sequence of TTL. The reduction in TTL activity is not accompanied by a decrease in the tyrosination levels of microtubules, as judged by immunofluorescence analysis. Strikingly, the number of cells in the plates transfected with the mutant TTL or the antisense TTL cDNA was, after 72 h of culture, two and three times higher, respectively, than the number of cells in the control plates. These results support the hypothesis that TTL may play a role in the regulation of the cell cycle in living cells.